Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme that has important roles in cell fate determination, and has been pursued as a therapeutic target. PARP-1 inhibitors have shown promise in selectively killing breast and ovarian cancers with certain DNA repair defects (BRCA1/2 negative). Difficult to treat cancers such as triple-negative breast cancer (TNBC) commonly fit this profile, presenting a significant potential and application of PARP-1 inhibitors. This example has introduced a new paradigm in chemotherapy termed synthetic lethality, where cancer cells with genetic defects are selectively killed with inconsequential effects on normal functioning cells. Current clinical trial are showing that patients with homologous recombination defects respond positively to PARP-1 inhibitors in monotherapy and in combination with traditional chemotherapeutic regimens. Currently, only one class of PARP-1 inhibitors has matured, although the development of alternative classes with improved selectivity is highly desirable. The recent structure discovery of key PARP-1 domains in complex with duplex DNA provides insights on how to develop new classes of PARP-1 inhibitors that can be more selective and effective. The objectives of this application are to (1) validate a new drug target site of PARP-1, (2) develop methods of detecting PARP-1 allosteric mechanisms and (3) identify small molecule scaffolds as new classes of PARP-1 inhibitors. A structure based drug design approach will be undertaken to test the hypothesis that development of alternative classes of PARP-1 inhibitors will diminish cross inhibition with other PARP isoforms and NAD binding enzymes, providing new tools that will broaden our understanding of PARP-1 with cancer and offer innovative cancer therapies. To test this hypothesis, the specific aims to be tested are (1) evaluation of the Zn3 domain of PARP-1 as a new drug target site, (2) development of high-throughput assays that are capable of detecting communication between PARP-1 domains upon DNA binding, and (3) to determine important structural interactions in the ADP-ribose pocket of PARP-1 that can assist a rational drug based design at this site. A combination of computational modeling and site-directed mutagenesis are being used to validate new drug target sites. In addition, two novel, complementary assays that can detect PARP-1 inter-domain communication upon activation are being designed. Crystallography with ligands constitutes a major component of this research plan, accelerating a structure based drug design approach. Identified inhibitors from this work are likely to be selective among PARP isoforms, allowing PARP-1 specific inhibition to be studied over multi-PARP inhibition. Long-term goals encompass development of new PARP-1 inhibitor candidates with translational potential that will be non-toxic and effective in treating cancers with BRCA1/2 and/or other DNA repair deficiencies.